Bottom Line:
While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching.Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity.Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.

Affiliation: W. R. Hearst Microbiology Research Center, Department of Microbiology &Immunology, Weill Medical College of Cornell University, New York, New York 10065, USA.

ABSTRACTEmerging evidence suggests that Cdc13-Stn1-Ten1 (CST), an RPA-like ssDNA-binding complex, may regulate primase-Pol α (PP) activity at telomeres constitutively, and at other genomic locations under conditions of replication stress. Here we examine the mechanisms of PP stimulation by CST using purified complexes derived from Candida glabrata. While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching. CST also simultaneously shortens the RNA and lengthens the DNA in the chimeric products. Stn1, the most conserved subunit of CST, is alone capable of PP stimulation. Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity. Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.

Figure 1: Characterization of the C. glabrata Pol α and the effect of CST on Pol α activity(a) C. glabrata PP (2 nM) was subjected to coupled primase-polymerase assays using the indicated ssDNA templates (poly-dT, at 300 nM, CgG4 and HsG9 at 1 µM). (b) PP (2 nM) was assayed using the CgG4 template and the indicated combinations of nucleotides. Total P32 incorporation into the products was quantified from PhosphorImger scans and plotted. Data (averages S.D.) are from three independent experiments. (c) The effect of CST (80 nM) on PP (1 nM) in the coupled primase-polymerase assays on poly-dT and CgG4 templates was analyzed. (d) The PP reaction products generated in the absence and presence of CST were subjected to alkaline hydrolysis and analyzed in a 13 % acrylamide-TBE-urea gel. The peak product length for each sample is indicated by a black dot to the right.

Mentions:
To investigate the regulation of PP by CST in vitro, we first isolated the two complexes encoded by C. glabrata. CST was obtained through recombinant co-expression and affinity purification from E. coli (Supplementary Fig. 1a)41, whereas PP through tagging of the Pri2 subunit and affinity purification from C. glabrata (Supplementary Fig. 1b). The PP complex can be further purified to near homogeneity by glycerol gradient fractionation (Supplementary Fig. 1c). The affinity-purified and glycerol gradient-purified PP behaved identically in all the polymerization assays used in the current study (Supplementary Fig. 1d). Before analyzing the effect of CST, we characterized the synthesis of RNA-DNA chimeras by PP alone on poly-dT and two model G-tail substrates (Fig. 1a and Supplementary Table 1). Physiologic concentrations of ribonucleotides and deoxyribonucleotides (including P32-dATP) were used in the assays to mimic the in vivo condition42. Because the synthesis of detectable products required the action of both primase and DNA polymerase, this assay will be referred to as the coupled primase-polymerase assay. Labeled products that ranged in size from about 10 nt to 40 nt were generated in these reactions. The product size distribution was quite narrow for the poly-dT template, manifesting a sharp peak at ~20–25 nt. In comparison, the C-strand products were more heterogeneous in size such that short (10–20 nt) and long (30–40 nt) products were as well represented as the medium-sized products (Fig. 1a). As predicted for PP-mediated synthesis of C-strand RNA-DNA chimeras, product accumulation required the presence both ribonucleotides and deoxyribonucleotides (Fig. 1b). Also consistent with the composition of the C. glabrata C-strand, omitting rCTP had more a detrimental effect than omitting other ribonucleotides on product synthesis. The different size distribution of the poly-dT and G-tail products indicates that the polymerization property of PP is sequence-dependent, as noted in a recent study43.

Figure 1: Characterization of the C. glabrata Pol α and the effect of CST on Pol α activity(a) C. glabrata PP (2 nM) was subjected to coupled primase-polymerase assays using the indicated ssDNA templates (poly-dT, at 300 nM, CgG4 and HsG9 at 1 µM). (b) PP (2 nM) was assayed using the CgG4 template and the indicated combinations of nucleotides. Total P32 incorporation into the products was quantified from PhosphorImger scans and plotted. Data (averages S.D.) are from three independent experiments. (c) The effect of CST (80 nM) on PP (1 nM) in the coupled primase-polymerase assays on poly-dT and CgG4 templates was analyzed. (d) The PP reaction products generated in the absence and presence of CST were subjected to alkaline hydrolysis and analyzed in a 13 % acrylamide-TBE-urea gel. The peak product length for each sample is indicated by a black dot to the right.

Mentions:
To investigate the regulation of PP by CST in vitro, we first isolated the two complexes encoded by C. glabrata. CST was obtained through recombinant co-expression and affinity purification from E. coli (Supplementary Fig. 1a)41, whereas PP through tagging of the Pri2 subunit and affinity purification from C. glabrata (Supplementary Fig. 1b). The PP complex can be further purified to near homogeneity by glycerol gradient fractionation (Supplementary Fig. 1c). The affinity-purified and glycerol gradient-purified PP behaved identically in all the polymerization assays used in the current study (Supplementary Fig. 1d). Before analyzing the effect of CST, we characterized the synthesis of RNA-DNA chimeras by PP alone on poly-dT and two model G-tail substrates (Fig. 1a and Supplementary Table 1). Physiologic concentrations of ribonucleotides and deoxyribonucleotides (including P32-dATP) were used in the assays to mimic the in vivo condition42. Because the synthesis of detectable products required the action of both primase and DNA polymerase, this assay will be referred to as the coupled primase-polymerase assay. Labeled products that ranged in size from about 10 nt to 40 nt were generated in these reactions. The product size distribution was quite narrow for the poly-dT template, manifesting a sharp peak at ~20–25 nt. In comparison, the C-strand products were more heterogeneous in size such that short (10–20 nt) and long (30–40 nt) products were as well represented as the medium-sized products (Fig. 1a). As predicted for PP-mediated synthesis of C-strand RNA-DNA chimeras, product accumulation required the presence both ribonucleotides and deoxyribonucleotides (Fig. 1b). Also consistent with the composition of the C. glabrata C-strand, omitting rCTP had more a detrimental effect than omitting other ribonucleotides on product synthesis. The different size distribution of the poly-dT and G-tail products indicates that the polymerization property of PP is sequence-dependent, as noted in a recent study43.

Bottom Line:
While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching.Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity.Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.

Affiliation:
W. R. Hearst Microbiology Research Center, Department of Microbiology &Immunology, Weill Medical College of Cornell University, New York, New York 10065, USA.

ABSTRACTEmerging evidence suggests that Cdc13-Stn1-Ten1 (CST), an RPA-like ssDNA-binding complex, may regulate primase-Pol α (PP) activity at telomeres constitutively, and at other genomic locations under conditions of replication stress. Here we examine the mechanisms of PP stimulation by CST using purified complexes derived from Candida glabrata. While CST does not enhance isolated DNA polymerase activity, it substantially augments both primase activity and primase-to-polymerase switching. CST also simultaneously shortens the RNA and lengthens the DNA in the chimeric products. Stn1, the most conserved subunit of CST, is alone capable of PP stimulation. Both the N-terminal OB fold and the C-terminal winged-helix domains of Stn1 can bind to the Pol12 subunit of the PP complex and stimulate PP activity. Our findings provide mechanistic insights on a well-conserved pathway of PP regulation that is critical for genome stability.